9 Jul

New publication from the lab: Cell stress induces chemoresistance

A combination of high-throughput analyses uncovers novel mechanism of stress-induced chemoresistance

Resistance of cancer cells against therapeutic agents is a major cause of treatment failure, especially in recurrent diseases. In a collaborative effort with the labs of Robert Ahrends, Björn Tews, Grischa Tödt and Christiane Knobbe-Thomsen, we identified a novel mechanism of chemoresistance which has now been published in ‘Nature Communications’. It is driven by the Unfolded Protein Response (UPR), a cellular stress response pathway that alters gene expression and cellular metabolism to promote cell survival under stress.


The Unfolded Protein Response (UPR), an important cellular stress response pathway, does not only contribute to cancer development, progression and chemoresistance, but also it plays an important role in numerous other diseases, among them diabetes and neurodegenerative disorders such as Alzheimer’s disease. A detailed biochemical understanding of the UPR is critically required to better define its role in disease and to develop novel therapeutic strategies. To produce a comprehensive description of the UPR, we employed a ‘multi-omics’ approach, combining large datasets from genetics and proteomics. This allowed us to define a list of genes (the UPR regulon) that are activated to promote cell survival under stress. Besides the previously known factors, we identified numerous genes that have not previously been implicated in stress response pathways and many of them have key functions in cancer development and cellular metabolism.

Schematic overview over the Unfolded Protein Response (UPR)

Changes in cellular metabolism are a hallmark of cancer cells and allow to sustain rapid tumor growth. Chemotherapy often aims at interfering with these metabolic pathways. We demonstrated that stress-mediated genetic regulation of enzymes involved in amino acid biosynthesis and one-carbon (1C) metabolism that relies on the vitamin folate as a cofactor. Moreover, upon stress, cancer cells become fully resistant to chemotherapeutic agents which target this specific metabolic pathway. This includes Methotrexate, a drug commonly employed in the treatment of cancer and rheumatic disease. Detailed biochemical and genetic investigations revealed that resistance is driven by a previously unrecognized mechanism. Its precise molecular characterization might lead to novel therapeutic concepts aimed at overcoming chemoresistance n cancer therapy.

Publication:

Reich S, Nguyen CDL, Has C, Steltgens S, Soni H, Coman C, Freyberg M, Bichler A, Seifert N, Conrad D, Knobbe-Thomsen CB, Tews B, Toedt G, Ahrends R, and Medenbach J: A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites – in Nature Communications, DOI:10.1038/s41467-020-16747-y

2 Mar

New publication: U2AF26 and U2AF35 as regulators of translation

A function in splicing versus a function in translational control – why not both?

U2AF proteins are best known for their functions in spliceosomal processing of pre-mRNAs where a homodimer of U2AF65 and U2AF35 functions in recognition of the 3′ splice site. The smaller subunit, U2AF35, contains two zinc fingers (ZnFs). Mutations therein have recently been associated with malignant transformation. The molecular function(s) of the two domains have, however, not been studied in great detail.

A collaborative effort, spearheaded by the Heyd lab at the Free University of Berlin, now revealed that the two ZnFs have remarkably different activity. Both are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65.

Intriguingly, a naturally occuring splice variant of U2AF26, a paralog of U2AF35, lacks the second ZnF. It is upregulated upon activation of primary mouse T cells and localizes to the cytoplasm, suggesting a splicing-independent function. Employing ribosome profiling in a model T cell line, we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increases translation when localized to the 5ʹUTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 in mammalian cells beyond the regulation of splicing.

Check out the exciting paper in RNA Biology: https://doi.org/10.1080/15476286.2020.1732701

16 Dec

New lab member: Andreas Meindl

Andreas Meindl has just joined the lab as a PhD student. Andreas will work on the function of Sex-lethal in Drosophila melanogaster sexual development addressing in detail its auto-regulatory feedback to splicing. Welcome to the lab!

2 Dec

Open position: Technical Assistant

You want to work on exciting and diverse research projects employing state-of-the-art methodologies? You want to join a young and highly motivated team? Then get your CVs ready, there is a job opening for a Technical Assistant!

The basic information: Salary TV-L E9, starting date as soon as possible, application deadline December 22nd 2019.

For more information (in German) please click here.

5 Nov

SFB960 PIs in 2019

The Collaborative Research Centre 960 (SFB960) ‘RNP biogenesis: assembly of ribosomes and non-ribosomal RNPs and control of their function’ has recently been awarded funding for another four years (see this post). The 18 group leaders (pictuerd below) will head 16 research and 3 service projects and a graduate school.

We are looking forward to four more years of collaborative research and exciting new findings.

Principal Investigators third funding period (from left to right): J. Medenbach, J. Griesenbeck, T. Heise, P. Milkereit, W. Seufert, A. Bruckmann, S. Ferreira-Cerca, M. Kretz, J. Perez-Fernandez, T. Dresselhaus, G. Längst, R. Sprangers, H. Tschochner, D. Grohmann, and C. Engel;
missing on the photo: G. Sommer, S. Sprunck, G. Meister, K. Grasser

14 Oct

New lab member: Mortiz Freyberg

Today, Moritz Freyberg joined the lab for his MSc work. He already spent some time with us for an extended practical and he will now continue to work on stress-mediated gene regulation in mammalian cells. Welcome back!

23 Jun

New publication: putting copy-numbers on UPR proteins

In a collaborative effort spearheaded by the Ahrends lab at the ISAS (Leibniz-Institut für Analytische Wissenschaften) in Dortmund, we established a targeted proteomics approach aimed at analyzing components of the Unfolded Protein Response (UPR), an adaptive signal transduction pathway triggered by the accumulation of unfolded proteins in the endoplasmic reticulum. The UPR comprises an important cellular stress response that aims at re-instating cellular homoeostasis and it plays a key role in a variety of disorders (including diabetes, neurodegenerative disorders, and inflammatory processes). It has also emerged as an attractive target for therapeutic intervention in cancer due to its implication in tumor progression, malignancy and resistance to therapy. The newly developed high-resolution targeted proteomics strategy combines high specificity and sensitivity, allowing the accurate quantification of UPR proteins down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. This has allowed us to determine cellular protein copy numbers of UPR receptors, transducers and effectors, yielding novel insights into an important cellular stress response pathway.

picture from: Nguyen et al., Scientific Reports, Volume 9, Article number: 8836 (2019) (CC BY 4.0)

 

Read the full manuscript at Scientific Reports: Nguyen et al. A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.

16 Jun

PhD positions available

We could successfully extend funding of the Collaborative Research Centre 960 (SFB960) ‘RNP biogenesis: assembly of ribosomal and non-ribosomal RNPs and control of their function’. To continue our ambitious research programs, we are now seeking highly motivated PhD students. We offer a highly competitive research environment and exciting research projects. For more information click here.

3 Jun

New publication from the lab – Auto-regulatory feedback by RNA-binding proteins

Mutations that alter the activity of RNA-binding proteins or their abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression.

Together with our colleagues Michaela Müller-McNicoll from the Institute of Cell Biology and Neuroscience at the Goethe University Frankfurt, Oliver Rossbach from the Institute of Biochemistry at the Justus-Liebig-University Giessen, and Jingyi Hiu at the State Key Laboratory of Molecular Biology (CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology), we have reviewed RBP-mediated autogenous feedback regulation in eukaryotic organisms. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression to either to maintain protein abundance within a physiological range (exerting negative feedback) or to enforce and stabilize cell fate decisions through generation of binary, genetic on/off switches.

The article has just been published in the Journal of Molecular Cell Biology – click here to read the full version.

23 May

The Regensburg Collaborative Research Centre SFB960 receives another four years of funding

Good news: the SFB 960 ‘RNP biogenesis: assembly of ribosomes and non-ribosomal RNPs and control of their function’ has been granted another funding period. We are grateful for the generous support by the Deutsche Forschungsgemeinschaft (DFG) that will allow us and our colleagues to continue and extend our scientific programs and to ensure further top-level education of PhD students within the Graduate Research Academy RNA Biology. We want to thank all reviewers that were involved in the selection process and we are looking forward to another four years of exciting science!

A news article on this topic can also be found on the homepage of the University of Regensburg (in German): DFG verlängert Regensburger Sonderforschungsbereich zur Ribosomen-Entstehung

10 May

New manuscript published – RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas

Picture from: Wirthschaft et al., Acta Neuropathol. 2019 (CC BY 4.0)

A collaborative effort lead by Björn Tews and supported by the research consortium ‘Systems Biology of the Unfolded Protein Response in Glioma’ (SUPR-G, generously funded by the BMBF in the framework of the e:med initiative) has resulted in a recent publication in Acta Neuropathologica that demonstrates a function of the peptide SPARC in migration and infiltrative growth of glioblastoma cells. SPARC production and secretion is enhanced via regulation of the UPR sensor IRE1 via AKT. SPARC secretion then prevents Nogo-A from inhibiting migration via RhoA. Advanced ultramicroscopy in undissected mouse brains reveals that gliomas require SPARC for invading into white matter structures and its depletion reduces tumor dissemination which significantly prolongs survival and improves response to cytostatic therapy. The discovery of a novel RhoA-IRE1 axis now provides a druggable target for interfering with SPARC production and underscores its therapeutic value. The full publiation can be accessed here.

15 Mar

New manuscript published – Purification of cross-linked RNA-protein complexes by phenol-toluol extraction (PTex)

We are happy that the collaborative effort spearheaded by Benedikt Beckmann at the Integrated Research Institute (IRI) for the Life Sciences has now resulted in a publication. We have described the approach earlier (see here) which, in a nutshell, allows the purification of cross-linked ribonucleoproteins by a series of organic extractions. Access the full article here at Nature Communications.

25 Jan

New publication from the lab: How to stabilize a sex-specific gene expression pattern in male flies

New manuscript from the lab published in Nucleic Acids Research: Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.

In a collboration with the labs of Stefan Schneuwly, Gunter Meister (both at the University of Regensburg), Michael Krahn (Westfälische Wilhelms-Universität Münster), and Oliver Rossbach (Justus-Liebig-University Giessen), we could demonstrate that the protein Sister-of-sex-lethal (Ssx) is required in male flies to suppress production of Sex-lethal (Sxl).

 

Genomic tagging of the Sex-lethal (Sxl) locus in flies to reveal Sxl protein mis-expression (arrowheads) in male flies mutant for Sister-of-Sex-lethal. Arrows mark expression of a Sxl isoform in neural cell bodies and projections.

 

Most higher eukaryotes reproduce sexually, increasing the variability in the offspring. This allows e.g. rapid adaption to a new (or changing) environment or the cleansing of harmful mutations from a population. Sexual reproduction in higher eukaryotes usually involves individuals of different sex: males and females. Not surprisingly, the genetic programs that determine sex and control sexual differentiation need to be particularly robust in order to ensure survival of the population.

In Drosophila, a single protein, the master regulator Sex-lethal (Sxl), governs female development by controlling the expression of key factors involved in female morphology and behaviour. Once expressed, it engages in an auto-regulatory, positive feedback loop to ensure its sustained expression. This stably ‘flips the switch’ and commits to female development.

In contrast, in males Sxl expression needs to be shut-off which is achieved by alternative splicing that generates RNA isoforms encoding truncated, non-functional Sxl protein. Fluctuations inherent to gene expression can, however, produce small amounts of functional Sxl protein. When left unchallenged, this protein can trigger a self-enforcing cascade resulting in Sxl protein expression snowballing out of control. Until now, however, it remained unclear how males completely shut off the Sxl expression cascade and protect themselves against runaway protein production to ensure robust sex-specific development.

We have discovered a safeguard mechanism that prevents Sxl production in adult male flies. We identified the protein Sister of Sex-lethal (Ssx) as the first antagonist of Sxl-mediated auto-regulatory splicing that defines a precise threshold level for activation of the auto-regulatory, positive feedback loop that controls Sxl expression. We could show that Ssx exerts function by competing with Sxl for the same RNA regulatory elements thus preventing Sxl from triggering the self-enforcing expression cascade in adult male animals.